FIG. 2A and FIG. 2B show a biosensor chip, as described in Hintsche, R., Paeschke, M., Uhlig, A., Seitz, R. (1997) “Microbiosensors using Electrodes made in Si-technology”, Frontiers in Biosensorics, Fundamental Aspects, Scheller, F W., Schubert, F., Fedrowitz, J. (eds.), Birkhauser Verlag Basle, Switzerland, pp. 267–283. The sensor 200 has two electrodes 201, 202 made of gold, which are embedded in an insulator layer 203 made of electrically insulating material. Connected to the electrodes 201, 202 are electrode terminals 204, 205, by means of which the electrical potential can be applied to the electrode 201, 202. The electrodes 201, 202 are configured as planar electrodes. DNA probe molecules 206 (also referred to as capture molecules) are immobilized on each electrode 201, 202 (cf. FIG. 2A). The immobilization is effected in accordance with the gold-sulfur coupling. The analyte to be investigated, for example an electrolyte 207 is applied on the electrodes 201, 202.
If the electrolyte 207 contains DNA strands 208 with a base sequence which is complementary to the sequence of the DNA probe molecules 206, i.e. which sterically match the capture molecules in accordance with the key/lock principle, then these DNA strands 208 hybridize with the DNA probe molecules 206 (cf. FIG. 2B).
Hybridization of a DNA probe molecule 206 and a DNA strand 208 takes place only when the sequences of the respective DNA probe molecule and of the corresponding DNA strand 208 are complementary to one another. If this is not the case, then no hybridization takes place. Thus, a DNA probe molecule having a predetermined sequence is in each case only capable of binding a specific DNA strand, namely the one with a respectively complementary sequence, that is to say of hybridizing with it, which results in the high degree of selectivity of the sensor 200.
If hybridization takes place, then the value of the impedance between the electrodes 201 and 202 changes, as can be seen from FIG. 2B. This changed impedance is detected by applying a suitable electrical voltage to the electrode terminals 204, 205 and by registering the current resulting from this.
In the case of hybridization, the impedance between the electrodes 201, 202 changes. This can be attributed to the fact that both the DNA probe molecules 206 and the DNA strands 208, which possibly hybridize with the DNA probe molecules 206, have poorer electrical conductivity than the electrolyte 207 and thus, as can be seen, in part electrically shield the respective electrode 201, 202.
In order to improve the measurement accuracy, it is known from van Gerwen, P. (1997) “Nanoscaled Interdigitated Electrode Arrays for Biochemical Sensors”, IEEE, International Conference on Solid-State Sensors and Actuators, Jun. 16–19 1997, Chicago, pp. 907–910, to use a plurality of electrode pairs 201, 202 and to arrange the latter in parallel with one another, these being arranged intermeshed with one another, as can be seen, so that the result is a so-called interdigital electrode 300, FIG. 3A showing the plan view thereof and FIG. 3B showing the cross-sectional view thereof along the section line I–I′ from FIG. 3A.
Furthermore, principles relating to a reduction/oxidation recycling process for registering macromolecular biomolecules are known for example from Hintsche et al., and Paeschke, M., Dietrich, F., Uhlig, A., Hintsche, R. (1996) “Voltammetric Multichannel Measurements Using Silicon Fabricated Microelectrode Arrays”, Electroanalysis, Vol. 7, No. 1, pp. 1–8. The reduction/oxidation recycling process, also referred to hereinafter as the redox recycling process, will be explained in more detail below with reference to FIG. 4A, FIG. 4B, FIG. 4C.
FIG. 4A shows a biosensor 400 having a first electrode 401 and a second electrode 402, which are applied on an insulator layer 403. A holding region 404 is applied on the first electrode 401 made of gold. The holding region 404 serves for immobilizing the DNA probe molecules 405 on the first electrode 401. Such a holding region is not provided on the second electrode 402.
If DNA strands 407 having a sequence which is complementary to the sequence of the immobilized DNA probe molecules 405 are intended to be registered by means of the biosensor 400, then the sensor 400 is brought into contact with a solution to be investigated, for example an electrolyte 406, in such a way that DNA strands 407 possibly contained in the solution 406 to be investigated can hybridize with the complementary sequence to the sequence of the DNA probe molecules 405.
FIG. 4B shows the case where the DNA strands 407 to be registered are contained in the solution 406 to be investigated and have hybridized with the DNA probe molecules 405.
The DNA strands 407 in the solution to be investigated are marked with an enzyme 408, with which it is possible to cleave molecules described below into partial molecules, at least one of which is redox-active. It is customary to provide a considerably larger number of DNA probe molecules 405 than there are DNA strands 407 to be determined contained in the solution 406 to be investigated.
After the DNA strands 407 possibly contained in the solution 406 to be investigated together with the enzyme 408 are hybridized with the immobilized DNA probe molecules 405, the biosensor 400 is rinsed, as a result of which the nonhybridized DNA strands are removed and the biosensor chip 400 is cleaned of the solution 406 to be investigated. The rinsing solution used for rinsing or a further solution supplied separately in a further phase has an electrically uncharged substance added to it, which contains molecules that can be cleaved by means of the enzyme 408 at the hybridized DNA strands 407, into a first partial molecule 410 and into a second partial molecule. One of the two molecules is redox-active.
As shown in FIG. 4C, the for example negatively charged first partial molecules 410 are attracted to the positively charged first electrode 401, which is indicated by means of the arrow 411 in FIG. 4C. The negatively charged first partial molecules 410 are oxidized at the first electrode 401, which has a positive electrical potential, and are attracted as oxidized partial molecules 413 to the negatively charged second electrode 402, where they are reduced again. The reduced partial molecules 414 again migrate to the positively charged first electrode 401. In this way, an electrical circulating current is generated, which is proportional to the number of charge carriers respectively generated by means of the enzymes 408.
The electrical parameter which is evaluated in this method is the change in the electric current m=dI/dt as a function of the time t, as is illustrated schematically in the diagram 500 in FIG. 5.
FIG. 5 shows the function of the electric current 501 depending on the time 502. The resulting curve profile 503 has an offset current Ioffset 504, which is independent of the temporal profile. The offset current Ioffset 504 is generated on account of non-idealities of the biosensor 400. An essential cause of the offset current Ioffset resides in the fact that the covering of the first electrode 401 with the DNA probe molecules 405 is not effected in an ideal manner, i.e. not completely densely. In the case of a completely dense coverage of the first electrode 401 with the DNA probe molecules 405, an essentially capacitive electrical coupling would result on account of the so-called double-layer capacitance, which is produced by the immobilized DNA probe molecules 405, between the first electrode 401 and the electrically conductive solution 406 to be investigated. However, the incomplete coverage leads to parasitic current paths between the first electrode 401 and the solution 406 to be investigated, which inter alia also have resistive components.
However, in order to enable the oxidation/reduction process, the coverage of the first electrode 401 with the DNA probe molecules 405 is intended not to be complete at all, in order that the electrically charged partial molecules, i.e. the negatively charged first partial molecules 410, can pass to the first electrode 401 on account of an electrical force and also as a result of diffusion processes. In order, on the other hand, to achieve the greatest possible sensitivity of such a biosensor, and in order simultaneously to achieve the least possible parasitic effects, the coverage of the first electrode 401 with DNA probe molecules 405 should be sufficiently dense. In order to achieve a high reproducibility of the measured values determined by means of such a biosensor 400, both electrodes 401, 402 are intended always to provide an adequately large area afforded for the oxidation/reduction process in the context of the redox recycling process.
Macromolecular biomolecules are to be understood for example as proteins or peptides or else DNA strands having a respectively predetermined sequence. If proteins or peptides are intended to be registered as macromolecular biomolecules, then the first molecules and the second molecules are ligands, for example active substances with a possible binding activity, which bind the proteins or peptides to be registered to the respective electrode on which the corresponding ligands are arranged.
Ligands that may be used are enzyme agonists, pharmaceuticals, sugars or antibodies or some other molecule which has the capability of specifically binding proteins or peptides.
If the macromolecular biomolecules used are DNA strands having a predetermined sequence which are intended to be registered by means of the biosensor, then it is possible, by means of the biosensor, for DNA strands having a predetermined sequence to be hybridized with DNA probe molecules having the sequence that is complementary to the sequence of the DNA strands as molecules on the first electrode.
A probe molecule (also called capture molecule) is to be understood as a ligand or a DNA probe molecule.
The value m=dI/dt introduced above, which corresponds to the gradient of the straight line 503 from FIG. 5, depends on the length and also the width of the electrodes used for registering the measurement current. Therefore, the value m is approximately proportional to the longitudinal extent of the electrodes used, for example in the case of the first electrode 201 and the second electrode 202 proportional to the length thereof perpendicular to the plane of the drawing in FIG. 2A and FIG. 2B. If a plurality of electrodes are connected in parallel, for example in the known interdigital electrode arrangement (cf. FIG. 3A, FIG. 3B), then the change in the measurement current is proportional to the number of electrodes respectively connected in parallel.
However, the value of the change in the measurement current may have a range of values that fluctuates to a very great extent, on account of various influences, the current range that can be detected by a sensor being referred to as the dynamic range. A current intensity range of five decades is often mentioned as a desirable dynamic range. Causes of the great fluctuations may be, in addition to the sensor geometry, also biochemical boundary conditions. Thus, it is possible that macromolecular biomolecules of different types to be registered will bring about greatly different ranges of values for the resulting measurement signal, i.e. in particular the measurement current and the temporal change thereof, which in turn leads to a widening of the required overall dynamic range with corresponding requirements for a predetermined electrode configuration with downstream uniform measurement electronics.
The requirements made of the large dynamic range of such a circuit have the effect that the measurement electronics are expensive and complicated in their configuration, in order to operate sufficiently accurately and reliably in the required dynamic range.
Furthermore, the offset current Ioffset is often much greater than the temporal change in the measurement current m over the entire measurement duration. In such a scenario, it is necessary, within a large signal, to measure a very small time-dependent change with high accuracy. This makes very high requirements of the measurement instruments used, which makes the registering of the measurement current complex, complicated and expensive. This fact is also at odds with a miniaturization of sensor arrangements that is striven for.
To summarize, the requirements made of the dynamic range and therefore of the quality of a circuit for detecting sensor events are extremely high.
It is known, during circuit design, to take account of the non-idealities of the components used (noise, parameter variations) in the form such that an operating point at which these non-idealities play a part that is as negligible as possible is chosen for these components in the circuit.
If a circuit is intended to be operated over a large dynamic range, maintaining an optimum operating point over all the ranges becomes increasingly more difficult, more complex and thus more expensive, however.
Small signal currents that are obtained at a sensor, for example, can be raised, with the aid of amplifier circuits to a level that permits the signal current to be forwarded for example to an external device or internal quantification.
A digital interface between the sensor and the evaluating system is advantageous for reasons of interference immunity and user-friendliness. Thus, the analog measurement currents are intended to be converted into digital signals actually in the vicinity of the sensor, which can be effected by means of an integrated analog-to-digital converter (ADC). Such an integrated concept for digitizing an analog small current signal is described in Uster, M., Loeliger, T., Guggenbühl, W., Jäckel, H. (1999) “Integrating ADC Using a Single Transistor as Integrator and Amplifier for Very Low (lfA Minimum) Input Currents”, Advanced A/D and D/A Conversion Techniques and their Applications, Conference at the University of Strathclyde (Great Britain) Jul. 27–28, 1999, Conference Publication No. 466, pp. 86–89, IEE, for example.
In order to achieve the required dynamic range, the ADC should have a correspondingly high resolution and a sufficiently high signal-to-noise ratio. Integrating such an analog-to-digital converter in direct proximity to a sensor electrode furthermore constitutes a high technological challenge, and the corresponding process implementation is complex and expensive. Furthermore, achieving a sufficiently high signal-to-noise ratio in the sensor is extremely difficult.